This invention in one aspect relates to a method for predicting and/or diagnosing diseases in living animals. The invention has particular utility in diagnosing and/or predicting future risk of specific diseases in living animals and will be described in connection with such utility, although other utilities are contemplated. This invention in another aspect relates to identification of markers for diseases or sub-clinical conditions that in the future may develop into diseases that are capable of distinguishing groups, and to subsets of these markers, where the utility of such markers can, for example, be determined by univariate, multivariate, or pattern recognition based analyses, and/or where the markers identified as important by the approach described also can be measured using other analytic approaches. The invention has particular applicability to predicting risk to cancer, type II diabetes, cardiovascular disease, cerebrovascular disease, and other diseases whose etiology has been established to or hypothesized to be modified by diet or nutrition, i.e. neurogenerative disorders such as Alzheimer""s Disease, Parkinson""s Disease and Huntington""s Disease {1}, and will be described specifically in connection its utility for using serum or plasma metabolites for determining breast cancer risk; however, other utilities and other tissue or biological fluid samples (e.g., whole blood, cerebrospinal fluid, urine, and/or tissue samples) may be used instead of blood, and diseases and conditions other than breast cancer also can be addressed, as noted above. Similarly, in addition to disease, the assessment of nutritive status (over long or short term), may be utilized in accordance with yet another aspect of the present invention as a medical test under a variety of potential clinical settings, or in controlling epidemiological or pharmaceutical testing. Still other utilities, e.g. for detecting exposure to and/or sensitivity to exposure to toxins, are contemplated.
Dietary restriction (DR), i.e. underfeeding without malnutrition, has established efficacy in reducing both degenerative and neoplastic diseases. DR has been extensively explored since its first use in the 1930""s because of its ability to extend both mean and maximum life span, reduce age-related morbidity, and delay or prevent certain age-associated physiological dysfunction {2, 3}. DR also alters many basic physiological processes, including metabolism, hormonal balance, and the generation of, detoxification of, and resistance to reactive oxygen species {4}. DR can be implemented in multiple ways {e.g. 5-13}. Moreover, restriction of total calories is believed to be more important than reducing intake of specific factors (e.g. fat, proteins, vitamins and minerals, etc. {14, 15}). DR reportedly extends longevity in essentially all animals in which it has been tried, including multiple mammalian species (rat, mouse, guinea pig {2, 5-13, 16}). Furthermore, promising data suggest that at least some of the benefits of DR, especially those regarding glucose metabolism, also occur in non-human primates {17-21}, and perhaps, in humans as well {22,23}. Together, these observations suggest that the DR effect is robust in mammals.
DR has been shown to reduce both incidence and severity of non-neoplastic diseases. One example is the efficacy of DR against glomerulonephritis, periarteritis, and myocardial degeneration in both male and female Sprague-Dawley rats. Similar observations have been made in other strains and other diseases, such as lung disease {25}. DR is also effective at preventing some strain specific disease, such as auto-immune disease in NZB/NZWF1 mice {26} and in MRL/1pr mice {27}, and atherosclerotic {28} and myocardial ischemia lesions in JCR:LA-cp mice {29}.
DR also has been shown to reduce both incidence and severity of neoplastic diseases. DR-mediated reduction of neoplasia includes delayed onset of leukemia, pituitary adenomas, mammary and prostatic tumors, and hepatomas {30, 31}. Observations of the effects of DR on mammary tumors {32-36} are typical. DR acts to reduce breast cancer both by delaying onset (both by reducing initiation events and slowing promotion) and by slowing tumor progression {30}. In transgenic mice prone to mammary tumors, DR reduced tumor incidence by 67% {32}. This result reveals that DR is capable of overcoming genetic predisposition to breast cancer. Studies {33} in rats treated with a carcinogen demonstrated that high fat and high calorie diets are co-carcinogenic, and that none of the rats maintained on 40% DR regimen developed mammary tumors, while 60% of AL-fed rats did. Concerns that this effect may have been partially mediated by reducing fat availability for tumor growth led to later studies {34}. Despite a higher fat content in the DR diet, results show a 75% reduction in rats with mammary tumors and in the number of tumors per animal in the tumor-bearing group. Even more impressively, DR reduced total tumor yield, average tumor size, and mean tumor burden by 93-98%. Notably, Sinha et al demonstrated that even a 20% DR regimen reduces tumors by 65%, without effects on hormone levels or fertility {35}.
Thus, DR mediated protection against breast cancer in laboratory models is: 1) substantial (as much as 100% reduction in cancer rates {32}) and highly replicable {30-34}; 2) robust and well-documented in a variety of animal models, including a model of genetic predisposition and a model of carcinogen exposure {31, 32}; 3) seen even with a more moderate (20%) restriction paradigm that does not affect fertility or hormone levels {34}; 4) effective at multiple levels (initiation, promotion, progression). Thus, the present invention, in one aspect, is based on the observation that different subsets of markers that reflect DR are predictive for different diseases. For example, identifying markers, for example in sera, that reflect the DR phenotype, would lead to markers that would reflect risk of developing breast cancer, or other conditions affected by diet.
Consistent with its broad effects on longevity and disease, DR is a systemic phenomenon, and its effects include measurable differences in blood constituents relative to those seen in ad libitum fed (AL) animals {37}. Many previous studies have focused on measurement of hormones. For instance, studies have shown alterations in plasma corticosterone patterns and levels {38}; some female reproductive hormones {39}, plasma chlecystokinin decreases 50% {40}; T3 but not T4 is reduced {41}; and plasma insulin drops as much as 60% in some DR models {42}. While informative, these studies have been somewhat limited by the technical complexity involved (e.g. circadian cyclicity, rapid response to stimuli). Other studies seeking more stable markers have examined markers of energy and free radical metabolism, revealing that DR decreases plasma glucose, ascorbate (e.g. 43-45) and glycohemoglobin levels {43}. Overall, the data indicates that differences in serotype distinguish AL and DR animals, and that these differences include some metabolites that are both relatively easy to assay and which reflect the beneficial effects of DR on physiology, metabolism and free radical biology (e.g. generation, sensitivity, and detoxification).
While not wishing to be bound by theory, since the AL and DR serotypes reflect robust physiological differences between these groups, it is believed that these serotypes include metabolites or metabolite profiles that cross-species and predict relative risk for the development of disease in humans. Data consistent with this concept comes from studies showing that the effect of DR on breast cancer is largely driven by chronic effects (termed promotion) rather than acute effects (termed initiation {30, 31}). These data would imply that relative risk of developing breast cancer is likely reflected in general metabolism over long periods of time. Relative risk should thus be detectable in sera long before the development of overt disease. In the case of humans, who lie on a broad spectrum with respect to caloric intake, it is believed that closer fit to the AL serotype (i.e. the biological response typical of a high caloric intake) would predict higher relative risk of disease, whereas greater fit to the DR serotype (i.e. the biological response typical of a lower caloric intake) would be associated with reduced risk. While previous studies demonstrated differences between AL and DR animals, they were believed only able to look at specific, predetermined markers, making it essentially impossible to conduct a sufficiently broad and powerful search to identify markers of use for determining nutritional status or predicting health across species.
The present invention provides a system, i.e. method and apparatus, for determining differences in concentrations of molecules, in particular small molecule metabolites, between animals whereby to create a metabolite database which may be used to reproducibly distinguish between two or more states of the health or the nutritive status of an animal. More particularly, the present invention employs analysis techniques to provide a small molecule inventory for metabolic pathway patterns of samples of ad libitum fed (AL) and dietary restricted (DR) individuals whereby to reproducibly distinguish between different dietary status of animals, between health conditions of animals, and to reproducibly predict relative risk for the development of a particular disease in animals.
The basis for this approach is that sufficient specific, reproducible, measurable changes exist in the overall biochemistry of small molecule metabolites among the different states to reproducibly distinguish the two (or more) states of interest. Different entities and/or sub-sets or combinations of markers can be used to identify different diseases or sub-clinical conditions. An HPLC-electrochemical analysis based approach in accordance with U.S. Pat. No. 4,863,873, which is incorporated herein by reference, has facilitated creation of a database for the constituents of AL and DR serum.